N-type calcium channel antagonists for the treatment of pain

ABSTRACT

Compounds useful for the treatment of pain in accord with the following structural diagram, 
                         
wherein A, R 1 , b, R 3 , d, R 4 , R 5 , R 6  and R 7  are any of a number of groups as defined in the specification, and pharmaceutical compositions and methods of treatment utilizing such compounds.

This is a 371 of International Application No. PCT/SE02/01521, filedAug. 23, 2002, which claims the benefit of Application No. 0102858-8filed in Sweden on Aug. 27, 2001.

FIELD OF THE INVENTION

This invention relates to substituted quinoline compounds, methods ofmaking such compounds and methods of using such compounds for thetreatment or prevention of pain or nociception.

RELATED ART

Pain causes a great deal of suffering and is a sensory experiencedistinct from sensations of touch, pressure, heat and cold. It is oftendescribed by sufferers by such terms as bright, dull, aching, pricking,cutting or burning and is generally considered to include both theoriginal sensation and the reaction to that sensation. This range ofsensations, as well as the variation in perception of pain by differentindividuals, renders a precise definition of pain difficult. Where painis “caused” by the stimulation of nociceptive receptors and transmittedover intact neural pathways, this is termed nociceptive pain. Pain mayalso be caused by damage to neural structures, and pain is often ismanifested as neural supersensitivity; this type of pain is referred toas neuropathic pain.

The level of stimulation at which pain is perceived is referred to asthe “pain threshold”. Where the pain threshold is raised, for instance,by the administration of an analgesic drug, a greater intensity or moreprolonged stimulus is required before pain is experienced. Analgesicsare a class of pharmaceutical agent which, following administration to apatient in need of such treatment, relieve pain without loss ofconsciousness. This is in contrast to other pain-relieving drugs, forexample, general anesthetics which obtund pain by producing a hiatus inconsciousness, or local anesthetics which block transmission inperipheral nerve fibers thereby preventing pain.

Tachykinin antagonists have been reported to induce antinociception inanimals, which is believed to be analogous to analgesia in man (forreview see Maggi et al, J. Auton. Pharmacol. 13:23–93 (1993)). Inparticular, non-peptide NK-1 receptor antagonists have been shown toproduce such analgesia, thus, for example, in classical tests ofchemo-nociception (phenylbenzoquinone-induced writhing and formalintest) the NK-1 receptor antagonist RP 67,580 produced analgesia withpotency comparable to that of morphine (Garret et al, Proc. Natl. Acad.Sci. USA 88:10208–10212 (1993)).

Opioid analgesics are a well-established class of analgesic agents.These compounds are generally accepted to include, in a generic sense,all drugs, natural or synthetic, with morphine-like actions. Thesynthetic and semi-synthetic opioid analgesics are derivatives of fivechemical classes of compound: phenanthrenes; phenylheptylamines;phenylpiperidines; morphinans; and benzomorphans. Pharmacologicallythese compounds have diverse activities, thus some are strong agonistsat the opioid receptors (e.g. morphine); others are moderate to mildagonists (e.g. codeine); still others exhibit mixed agonist-antagonistactivity (e.g. nalbuphine); and yet others are partial agonists (e.g.nalorphine). Whilst an opioid partial agonist such as nalorphine, (theN-alkyl analogue of morphine) will antagonize the analgesic effects ofmorphine, when given alone it can be a potent analgesic in its ownright. Of all of the opioid analgesics, morphine remains the most widelyused and is a suitable archetype compound. Unfortunately, apart from itsuseful therapeutic properties, morphine also has a number of drawbacksincluding respiratory depression, decreased gastrointestinal motility(resulting in constipation) and, in some individuals, nausea andvomiting may occur. Another characteristic is the development oftolerance and physical dependence which may limit the clinical use ofsuch compounds.

Anti-inflammatory compounds directed at blocking or reducing synovialinflammation, and thereby improving function, and analgesics directed toreducing pain, are presently the primary method of treating therheumatoid diseases and arthritis. Aspirin and other salicylatecompounds are frequently used in treatment to interrupt amplification ofthe inflammatory process and temporarily relieve the pain. Other drugcompounds used for these purposes include phenylpropionic acidderivatives such as Ibuprofen and Naproxin, Sulindac, phenyl butazone,corticosteroids, antimalarials such as chloroquine andhydroxychloroquine sulfate, and fenemates. For a thorough review ofvarious drugs utilized in treating rheumatic diseases, reference is madeto J. Hosp. Pharm., 36:622 (May 1979).

Calcium channels are membrane-spanning, multi-subunit proteins thatallow controlled entry of Ca⁺⁺ ions into cells from the extracellularfluid. Such channels are found throughout the animal kingdom, and havebeen identified in bacterial, fungal and plant cells. Commonly, calciumchannels are voltage dependent. In such channels, the “opening” allowsan initial influx of Ca⁺⁺ ions into the cells which lowers the potentialdifference between the inside of the cell bearing the channel and theextracellular medium bathing the cell. The rate of influx of Ca⁺⁺ ionsinto the cell depends on this potential difference. All “excitable”cells in animals, such as neurons of the central nervous system (“CNS”),peripheral nerve cells, and muscle cells, including those of skeletalmuscles, cardiac muscles, and venous and arterial smooth muscles, havevoltage-dependent calcium channels. Calcium channels are physiologicallyimportant because the channels have a central role in regulatingintracellular Ca⁺⁺ ions levels. These levels are important for cellviability and function. Thus, intracellular Ca⁺⁺ ion concentrations areimplicated in a number of vital processes in animals, such asneurotransmitter release, muscle contraction, pacemaker activity, andsecretion of hormones.

It is believed that calcium channels are relevant in certain diseasestates. A number of compounds useful in treating various cardiovasculardiseases in animals, including humans, are thought to exert theirbeneficial effects by modulating functions of voltage-dependent calciumchannels present in cardiac and/or vascular smooth muscle. Many of thesecompounds bind to calcium channels and block, or reduce the rate of,influx of Ca⁺⁺ ions into the cells in response to depolarization of thecell membrane. An understanding of the pharmacology of compounds thatinteract with calcium channels in other organ systems, such as thecentral nervous system, and the ability to rationally design compoundsthat will interact with these specific subtypes of human calciumchannels to have desired therapeutic, e.g., treatment ofneurodegenerative disorders, effects have been hampered by an inabilityto independently determine how many different types of calcium channelsexist or the molecular nature of individual subtypes, particularly inthe CNS, and the unavailability of pure preparations of specific channelsubtypes, i.e., systems to evaluate the specificity of calciumchannel-effecting compounds.

Multiple types of calcium channels have been detected based onelectrophysiological and pharmacological studies of various mammaliancells from various tissues (e.g., skeletal muscle, cardiac muscle, lung,smooth muscle and brain) Bean, B. P., Annu. Rev. Physiol. 51:367–384(1989) and Hess, P., Annu. Rev. Neurosci. 56:337 (1990). These differenttypes of calcium channels have been broadly categorized into fourclasses, L-, T-, N-, and P-type, distinguished by current kinetics,holding potential sensitivity and sensitivity to calcium channelagonists and antagonists. Four subtypes of neuronal voltage-dependentcalcium channels have been proposed Swandulla, D. et al, Trends Neurosci14:46 (1991). The L-, N-, and P-type channels have each been implicatedin nociception, but only the N-type channel has been consistentlyimplicated in acute, persistent and neuropathic pain. A syntheticversion of ω-conotoxin MVIIA, a 25-amino acid peptide derived from thevenom of the piscivorous marine snail, Conus magus has been usedintrathecally in humans and has ˜85% success rate for the treatment ofpain with a greater potency than morphine.

While known drug therapies have utility, there are drawbacks to theiruse. For instance, it may take up to six months of consistent use ofsome medications in order for the product to have effect in relievingthe patient's pain. Consequently, a particular subject may be receivingtreatment and continuing to suffer for up to six months before thephysician can assess whether the treatment is effective. Many existingdrugs also have substantial adverse side effects in certain patients,and subjects must therefore be carefully monitored. Additionally, mostexisting drugs bring only temporary relief to sufferers and must betaken consistently on a daily or weekly basis for continued relief.Finally, with disease progression, the amount of medication needed toalleviate the pain may increase thus increasing the potential for sideeffects. Thus, there is still a need for an effective and safe treatmentto alleviate pain.

SUMMARY OF THE INVENTION

In one aspect the present invention provides compounds having selectiveaction at N-type calcium channels that are useful for the treatment ofpain.

Compounds of the present invention that show selective action at N-typecalcium channels are compounds in accord with structural diagram I,

wherein:

A is selected from phenyl, heteroaryl or bicyclic heteroaryl;

R¹ at each occurrence is independently selected from halogen,(C₁–C₆)alkyl, heterocyclyl, OH, (C₁–C₆)alkoxy, or NR² ₂;

b is an integer selected from 0, 1, 2 or 3;

R² at each occurrence is independently selected from H or (C₁–C₄)alkyl;

R³ at each occurrence is independently selected from halogen or(C₁–C₄)alkyl;

d is an integer selected from 0, 1, 2 or 3;

R⁴ is selected from H or (C₁–C₄)alkyl;

R⁵ is selected from the group consisting of H, halogen, (C₁–C₃)alkyl,(C₁–C₃)perfluoroalkyl, (C₁–C₃)alkoxy, hydroxy, NH₂ or NHR²;

R⁶ is selected from the group consisting of H, halogen, (C₁–C₆)alkyl,(C₁–C₄)perfluoroalkyl, (C₁–C₆)alkoxy, hydroxy, (C₁–C₆)alkanoyl, C(═O)NR²₂ or NR² ₂; and

R⁷ is selected from H or methyl.

Particular compounds of the present invention are those in accord withstructural diagram I wherein;

A is selected from phenyl or bicyclic heteroaryl;

R¹ at each occurrence is independently selected from halogen,(C₁–C₆)alkyl, heterocyclyl, OH, (C₁–C₆)alkoxy, or NR² ₂;

b is an integer selected from 0, 1, 2 or 3;

R² at each occurrence is independently selected from H or (C₁–C₄)alkyl;

R³ at each occurrence is independently selected from halogen or(C₁–C₄)alkyl;

d is an integer selected from 0, 1, 2 or 3;

R⁴ is H;

R⁵ is selected from H, halogen, (C₁–C₃)alkyl, (C₁–C₃)perfluoroalkyl or(C₁–C₃)alkoxy;

R⁶ is selected from H, halogen, (C₁–C₆)alkyl, (C₁–C₄)perfluoroalkyl,(C₁–C₆)alkoxy, hydroxy, (C₁–C₆)alkanoyl, C(═O)NR₂ or —NR²; and

R⁷ is H.

Most particular compounds of the invention are those exemplified herein.

In another aspect, the invention comprises a method for using compoundsaccording to structural diagram I for the treatment of pain, said methodcomprising administering a pain-ameliorating effective amount of anysuch compound.

One embodiment of the method of the invention comprises administering apain-ameliorating effective amount of a compound in accordance withstructural diagram I to a subject in need of treatment for acute,persistent or neuropathic pain.

In a further aspect, the invention comprises methods for makingcompounds in accord with structural diagram I.

In yet another aspect, the invention comprises pharmaceuticalcompositions comprising compounds in accord with structural diagram Itogether with excipients, diluents or stabilizers, as further disclosedherein, useful for the treatment of acute, persistent and neuropathicpain.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the invention are those within the scope of the genericdescription and particularly those compounds exemplified hereafter.

Suitable pharmaceutically-acceptable salts of compounds of the inventioninclude acid addition salts such as methanesulfonate, fumarate,hydrochloride, hydrobromide, citrate, tris(hydroxymethyl)aminomethane,maleate and salts formed with phosphoric and sulfuric acid.

Where compounds of the present invention possess a chiral center it isto be understood that the invention encompasses all optical isomers anddiastereoisomers of such compounds.

Where compounds of the present invention can tautomerize it is to beunderstood that the invention encompasses all tautomeric forms of suchcompounds.

Where compounds of the present invention can exist in unsolvated as wellas solvated forms such as, for example, hydrated forms, it is to beunderstood that the invention encompasses all such solvated andunsolvated forms.

Another aspect of the invention provides processes for making compoundsof the invention. Generally compounds of the present invention wereprepared by reacting in an automated fashion substituted2-chloro-4-aminoquinoline intermediates with aminoaryl precursors. Theintermediate substituted 2-chloro-4-aminoquinolines were prepared bychlorination of substituted 2-hydroxy-4-aminoquinoline precursors, whichwere in turn prepared by amination of substituted 2,4-quinolinediolprecursors.

-   a) 2-Hydroxy-4-aminoquinoline precursors in accord with structural    diagram III were prepared by reacting a substituted    2,4-quinolinediol in accord with structural diagram II with three    equivalents of an aryl amine in N-methylpyrrolidinone and 6N HCL in    2-propanol, in a sealed tube at a temperature of about 180° C.

wherein R³, R⁴, R⁵, R⁶ and d, are as heretofore defined;

-   a′) 2-hydroxy-4-aminoquinoline precursors in accord with structural    diagram III were alternatively prepared by reacting a substituted    2,4-quinolinediol in accord with structural diagram II with two    equivalents of an aryl amine in N-methylpyrrolidinone and 4N HCL in    dioxane, in a sealed teflon tube. The reaction was maintained at a    temperature of about 200° C. using an Ethos 1600 Lab Microwave as    the energy source.-   b) 2-chloro-4-aminoquinoline precursors were prepared by    chlorinating a 2-hydroxy-4-aminoquinoline compound in accord with    structural diagram III by refluxing with POCl₃ to form a compound    according to structural diagram IV.

wherein R³, R⁴, R⁵, R⁶, b and d are as heretofore defined;

-   c) Compounds of the present invention in accord with structural    diagram I were prepared by reacting 2-chloro-4-aminoquinoline    precursors with aromatic amines in N-methylpyrrolidinone at    temperatures of 100–180° C.

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, b and d are as heretofore defined.

The procedure of step c) may also be performed in a parallel fashionusing a robotic instrumentality. A suitable robotic instrumentality forsuch a multiple parallel synthesis is a ChemSpeed robot.

To use a compound of the invention or a pharmaceutically-acceptable saltthereof for the therapeutic treatment or prophylactic treatment of painin mammals, which may be humans, the compound can be formulated inaccordance with standard pharmaceutical practice as a pharmaceuticalcomposition. Accordingly, a further aspect of the invention provides apharmaceutical composition which contains a compound of the structuraldiagram I as defined herein or a pharmaceutically-acceptable saltthereof, in association with at least one pharmaceutically-acceptableadditive such as an excipient or carrier.

In methods of the invention, treatment is contemplated to beadministered in any physiologically-acceptable, such as by topicalapplication, ingestion, inhalation insufflation or injection. Topicalapplication, for example may be by a dermal, sublingual, nasal, vaginalor rectal route. Injection may be intradermal, subcutaneous, parenteral,intraperitoneal, intravenous, intramuscular or by infusion. Ingestionmay be of a capsule a tablet, or a liquid. Suitable pharmaceuticalcompositions that contain a compound of the invention may be formulatedby means known in the art in the form of, for example, tablets,capsules, aqueous or oily solutions or suspensions, emulsions, creams,ointments, gels, nasal sprays, suppositories, finely divided powders oraerosols for inhalation, and for injection sterile aqueous or oilysolutions or suspensions or sterile emulsions. A preferred route ofadministration is orally by tablet or capsule.

In addition to a compound of the present invention, a pharmaceuticalcomposition of this invention may also contain one or more otherpharmacologically-active agents. Alternatively, a pharmaceuticalcomposition comprising a compound of this invention may beco-administered simultaneously or sequentially with one or more othercompatible pharmacologically-active agents.

Pharmaceutical compositions of this invention will normally beadministered so that a pain-ameliorating effective daily dose isreceived by the subject. The daily dose may be given in divided doses asnecessary, the precise amount of the compound received and the route ofadministration depending on the weight, age and sex of the patient beingtreated and on the particular disease condition being treated accordingto principles known in the art. A preferred dosage regime is once daily.

A yet further embodiment of the invention provide the use of a compoundof the structural diagram I, or a pharmaceutically-acceptable saltthereof, in the manufacture of a medicament useful for binding to N-typecalcium channels in a warm-blooded animal such as a human being.

Still another embodiment of the invention provides a method of binding acompound of the invention to N-type calcium channels of a warm-bloodedanimal, such as a human being, in need of treatment for pain, whichmethod comprises administering to said animal an effective amount of acompound of structural diagram I or a pharmaceutically-acceptable saltthereof.

A further aspect of the present invention provides a pharmaceuticalcomposition which includes a compound of the present invention asdefined herein or a pharmaceutically-acceptable salt thereof, inassociation with a pharmaceutically-acceptable additive such as anexcipient or a carrier.

A still further aspect of the present invention is a method of treatmentof the human or animal body that includes the administration of acompound of the present invention or a pharmaceutically-acceptable saltthereof.

DEFINITIONS

When used herein “halo” or “halogen” means fluoro, chloro, bromo oriodo;

when substituents herein are stated to be “selected from” or“independently selected from” a group of moieties, it is to beunderstood that included compounds are those where all substituents arethe same and compounds where each substituent may be different;

when used herein, the term heterocyclyl includes 5–7 membered ringscontaining 1, 2 or 3 heteroatoms selected from the group consisting ofN, O and S and bicyclic rings containing such atoms and includes suchgroups as tetrahydrofuryl, dihydropyrrolynyl, tetrahydroisoquinolinyl,tetrahydrothiophene, oxiranyl, azidiridynl and oxetanyl

when used herein, the term heteroaryl includes such groups as pyridinyl,pyrrole, thiophenyl and furanyl;

when used herein the term “alkyl,” as in for example (C₁–C₆)alkyl,unless otherwise defined, includes both straight, branched and cyclicchain alkyl groups. References to individual alkyl groups such as“propyl” mean the normal, straight chain form, that is, n-propyl;

when used herein, a term such as “(C₁–C₆)alkyl” means alkyl groupshaving 1, 2, 3, 4, 5 or 6 carbon atoms and collective groups such as(C₁–C₄)alkyl and includes straight, branched and cyclic moieties,straight chain moieties include for example methyl, ethyl and propyl,branched chain moieties include for example iso-propyl and t-butyl,cyclic moieties include for example cyclopentyl and cyclopropylmethyl;similarly, a term such as “(C₁–C₃)alkoxy” includes particular moietiessuch as methoxy, ethoxy and propoxy, and terms used herein that are nototherwise defined are intended to have their conventionally-understoodmeaning.

The Methods and Examples which follow are intended to illustrate but notlimit the invention. In the Methods and Examples, unless otherwisestated:

concentrations of solutions were carried out by rotary evaporation invacuo;

operations were carried out at ambient temperature, that is in the range18–26° C. and under a nitrogen atmosphere;

column chromatography (by the flash procedure) was performed on MerckKieselgel silica (Art. 9385);

yields are given for illustrative purposes only and are not necessarilythe maximum attainable;

the structure of compounds according to structural diagram I weregenerally confirmed by conventional NMR (Bruker Avance 300) and massspectrometry techniques, peak multiplicities are shown thus: s, singlet;bs, broad singlet; d, doublet; AB or dd, doublet of doublets; t,triplet; dt, double of triplets; m, multiplet; bm, broad multiplet; FABm/s data were obtained using a Platform spectrometer (supplied byMicromass) run in electrospray and, where appropriate, either positiveion data or negative ion data were collected, herein (M+H)⁺ is quoted;

purity of intermediates were was in general assessed by LC/MS and/or NMRanalysis; and where used the following abbreviations have meanings asfollows:

DCM is dichloromethane, DMF is N,N-dimethylformamide, DMSO isdimethylsulfoxide, CDCl₃ is deuterated chloroform, FAB is fast atombombardment, LC/MS is mass spectrometry linked to liquid chromatographyinstrumentation m/s is mass spectroscopy or mass spectral, NMR isNuclear Magnetic Resonance, NMP is N-methylpyrrolidinone, TFA istrifluoroacetic acid, and THF is tetrahydrofuran.

BIOLOGICAL METHODS

I. N-channel FLIPR (Fluorescent Laser Imaging Plate Reader) Assay

The methods described herein provide a reliable FLIPR-based readout ofthe efficacy and potency with which test compounds inhibit calcium fluxthrough the N-type calcium channel expressed in its native form in ahuman-derived neuroblastoma cell line differentiated chemically to aneuronal phenotype. The degree to which a compound at a particularconcentration inhibited the N-channel calcium flux was determined bycomparing the amplitude of peak calcium increase in the presence of thecompound to a control 80 mM K⁺ stimulus in wells without compound.Results obtained for this FLIPR assay were validated in two ways:

a) the N-channel specific peptide toxin, conotoxin MVIIA, showed anIC₅₀=3 nM (determined from fit to five-point concentration responseanalysis), compatible with the known literature value; and

b) IC₅₀ values were determined certain compounds of the invention (IC₅₀range: 2.37–10.54).

Potency of these same test compounds as inhibitors of the N-type calciumcurrent was also determined by direct electrophysiological measurementeither in neuronally differentiated IMR-32 cells, or in freshly-isolatedrat superior cervical ganglion neurons pIC₅₀'s yielded by the twomethodologies for the compound set were closely comparable (r=0.91;p<0.001).

A. Cell Culture

An immortalized cell line, IMR32, derived from human neuroblastoma cellsobtained from the ATCC (product #CCL-127) was used for all experiments.Cells were grown in T75 flasks containing Eagle's minimum essentialmedium (MEM) w/Earle's salts and non-essential amino acids withoutglutamine (Cat.#SLM-034-B, Specialty Media, Philipsburg, N.J.), 10% FBSand 1% glutamine. Cells were grown to ˜70–80% confluency (by visualmicroscopic estimation) before sub-culturing. To maintain a stockculture, cultures were split at a ratio of 1:3–1:4 by creating a cellsuspension by trituration, and pipetting a volume of the cell suspensionsufficient to yield this final ratio into new flasks containing ˜20 mLof fresh media. Sub-culturing was generally performed two times perweek. For preparation of 96 well plates (black-walled; Cat # 3603,Costar Co., Cambridge, Mass.), a T75 flask containing cells of desiredconfluency was brought up to 120 mL volume with media. Cells were thenfreed by trituration, and the cell suspension was plated into 12–96 wellplates to yield final well volume of 100 μL.

B. Cell Differentiation to Neuronal Phenotype

Cells were induced to differentiate in a differentiation mediumconsisting of: MEM, 10% FBS, 1% glutamine, 1 μM 2-butyl-cAMP(49.1 mg/100mL media(Cat. # D-0627, Sigma Corp., St Louis, Mo.), and 2.5 nMbromo-deoxy-uridine (stock: 30.7 mg/10 mL media, 25 mL of abovestock/100 mL media; Sigma Cat .# B-9285). To induce differentiation, thecells were treated with differentiation media (by complete mediumchange) 2 days after an initial plating in 96 well plates. Confluency atthis time was ˜40%. A complete medium change with freshly prepareddifferentiating medium was subsequently performed every 2–3 days. Cellswere exposed to these differentiation conditions for 6 to 11 days beforebeing used in FLIPR experiments.

C. Standard Experimental Solutions

Solutions of the following composition (in nM) were used in experiments(Buffers without probenicid purchased from Specialty Media (Buffers Aand B: Cat. # BSS053A; Buffers C & D: Cat. # BSS056A).

Buffer A (first wash buffer): Krebs-Ringer-HEPES (KRH) buffer: NaCl:125, KCl: 5, MgSO₄: 1.2, KH₂PO₄: 1.2, CaCl₂2H₂O: 2, Glucose: 6, HEPES:25, pH: 7.4 (pH adjusted with NaOH).

Buffer B (dye loading buffer) KRH buffer with 2.5 μM probenicid: same asbuffer A, but probenicid added to final concentration of 2.5 μM.Probenecid (Cat. # P-8761, Sigma Chemical Co., St. Louis, Mo.) made as astock solution at 250 mM.

Buffer C (dye washout buffer) KRH buffer with 0 mM K⁺ and 2.5 μMprobenicid: NaCl: 130, MgSO₄:1.2, NaH₂PO₄: 1.2, CaCl₂ 2H₂O: 2, Glucose:6, HEPES: 25, pH: 7.4 (pH adjusted with NaOH).

Buffer D (compound dilution buffer). Buffer C with 0.1% w/v bovine serumalbumin (BSA; Sigma).

D. Pharmacological Standards and Compounds

The following solutions were used to obtain the data disclosed herein.

Nitrendipine: (RBI Chemicals, Natick, Mass.): Stock: 10 mM in DMSO;Pipetting solution: 9 μM; pipette 20 μL into 120 μL volume in well forfinal well concentration: 1 μM.

w-Conotoxin MVIIA: (Cat. # H-8210; Bachem Inc., Torrance, Calif.):Stock: 1 mM in HPLC grade H₂ 0 with 0.1% BSA; Pipetting solution: 4.5μM; pipette 20 μl into 140 μl volume in well for final wellconcentration: 1 μM.

Test compound stock and solution preparation: Compounds prepared dailyas stocks at 10 mM in 100% DMSO; Pipetting solution: 45 μM or serialdilutions thereof; pipette 20 μL into 140 μL volume in well for finalwell concentration: 1 μM or 10-fold dilutions thereof.

High potassium (depolarization) solution: Buffer C with 240 mM K⁺ added;pipette 80 μL into 160 μL volume in well for final well concentration of80 mM K⁺.

E. Cell Loading with Fluorescent Dyes

Fluorescent dye solution preparation: A calcium indicator dye, Fluo-4acetylmethylester (Fluo 4-AM; Cat. # F-124201; Molecular Probes, Eugene,Oreg.) was used to measure changes in intracellular free calcium withFLIPR. 1 mM Fluo-4 μM stock solution was made by dissolution in DMSO.This stock was then diluted to 4.6 μM with Buffer B (Fluo-4 AM workingsolution).

Cell loading procedure: Plates containing cells were washed with BufferA using an automated cell washer (Model #: 5161552, Labsystems Oy,Helsinki, Finland) with controls set to the following parameters: cellheight: C/D; cell pulse: 4/5, washes: 3; volume: 5; DRY positionsetting. These settings resulted in a 70 μL residual depth of bufferover cells in each well. 100 μL of the Fluo-4 AM working solution wasthen added to each well resulting in a final Fluo-4 AM concentration of2.7 μM Cells were incubated in this solution at 37° C. for 1–1.5 h.Cells were then washed with Buffer C five times using the cell washerwith parameters the same as the pre-loading washes above with theexceptions of: washes: 5; WET position setting. A final wash was thenconducted by changing the parameters as follows: washes: 1; volume: 2.This resulted in a final well volume of 120 μL. Cells were allowed toequilibrate under this condition for 10 min, and then used in the FLIPRprotocol.

F. FLIPR Protocol

Instrumentation: Real time changes in intracellular free calcium inresponse to potassium-induced depolarization in the absence or presenceof putative N-channel inhibitors were measured by either a FLIPR I orFLIPR II (configured for 96-well format) instrument (Molecular Devices,Sunnyvale, Calif.). Identical settings and protocols were used with eachinstrument, and results obtained from the two instruments wereindistinguishable for a set of standard benchmark compounds.

FLIPR hardware settings: Laser power was set to about 0.3 watts.Excitation wavelength was set to a 488 nm peak, and the emissionwavelength to 540 nm. Camera aperture was set to 2. All experiments wereconducted at room temperature (20–22° C.).

Plate layout—reference signals: Certain wells on each plate wereallocated to standards to determine minimum and maximum specificfluorescent signal against which inhibitory effects of compounds werenormalized. The reference standards were distributed at plate locationsincluding edge and interior wells.

Maximum signal (N-channel+non-specific): 12 wells were incubated innitrendipine (1 μM) solution and 80 mM K⁺ added to determine maximalCa²⁺ increase mediated by N-channels+non-specific (non-L-, non-N-channelmediated fluorescence increase). The coefficient of variation amongstthese wells for the K⁺-evoked peak increase in fluorescence units wastypically less than 12%.

Minimum signal (non-specific): 6 wells were incubated in nitrendipine (1μM)+w-conotoxin MVIIA and 80 mM K⁺ added to determine background Ca²⁺with all N-channels pharmacologically occluded The peak non-specificsignal component was typically less than 15% of the maximum signal peakamplitude.

N-channel reference small molecule: A compound that had beencharacterized extensively with respect to N-channel inhibitory activityin both FLIPR and patch clamp electrophysiology was included on eachplate in triplicate at 1 μM (near IC₅₀) to establish a reference point.

Test compounds: 5 test compounds were evaluated for potency on eachplate. Each compound was tested at 5 increasing concentrations spanninghalf-log units and typically reaching a maximal concentration of 10 μM.Each concentration was tested in triplicate wells.

Protocol structure: The FLIPR protocol was configured as three solutionaddition/sampling sequences (see below). Conotoxin (1 μM final conc.)was added to appropriate wells prior to placing the plate in the FLIPRinstrument. Wells initially contained a total solution volume of 100 μl,and after all three solution additions contained 240 μl. The activemixing (by the pipette) option was not used in any sequence.

Nitrendipine addition sequence: 28 s total duration with fluorescencesignal sampling at 1 Hz for 2 s, followed by addition of 20 μLnitrendipine standard solution at 10 μL/s, followed by sampling at 0.5Hz for 24 s.

Test compound addition sequence: 64 s total duration with sampling at0.5 Hz for 4 sec, test solution addition of 40 μL at 20 μL/s, followedby sampling at 0.2 Hz for 60 s.

Compound incubation, cell depolarization and calcium readout sequence:1024 s total duration with sampling at 0.0167 Hz for 840 s, followed bysolution addition 80 μL of high K⁺ (depolarization) solution, followedby sampling at 1 Hz for 180 sec. This final 180 sec sampling intervalthus represented the epoch where the peak increase in intracellularcalcium due to flux through activated N-channels occurred.

G. Data Analysis

FLIPR software: Prior to export, the data was normalized within theFLIPR software module for two effects.

Baseline correction: The baseline was corrected by “zeroing” at sample #57 (immediately prior to KCl addition). This normalization served tocorrect the y axis offset of the fluorescent trace from each well sothat all traces had a common point just prior to onset of the relevantevoked fluorescent increase.

Spatial uniformity correction factor: The data was normalized by aprocedure which calculates a mean over the plate of fluorescent unitsfrom the first sample, and then multiplies the data from each well by ascalar that adjusts the value of the first sample to this average value,thus normalizing for differences in absolute baseline fluorescenceamongst the wells caused by differences in cell densities or dyeloading.

External software: Data were exported from FLIPR into Excel as “*.squ”extension files. Following export, operations were performed in Excel tocalculate the maximal peak amplitude (relative to the zeroed baseline)of the fluorescence increase following potassium addition in each well.Measurements from wells where an test compound was added were thennormalized as a percentage between the mean amplitudes from thereference wells providing the maximum (100%) and non-specific (0%)signal components, as described above. The resulting percent inhibitionby test compounds was considered to reflect inhibition of calcium fluxat the N-type channel.

II. Formalin Test

The Formalin test assesses the inhibitory effects of orally administeredN-type calcium channel antagonists on formalin-induced nocifensivebehaviours in rats. The formalin test is a well established pain test(Dubuisson and Dennis, 1977; Wheeler-Aceto et al., 1990; Coderre et al.,1993). This test consists of two distinct phases of formalin-inducedbehavior. The first phase response, occurring between 0 to 5 minutes, iscaused by acute nociception to the noxious chemical (formalin) injectedinto the paw. This is followed by a quiescent period of between 5 to 15min post injection. A second phase response, occurring after 15 minutesand lasting up to 60 minutes, is caused by sensitization of the centralneurons in the dorsal horn. Central sensitization augments the noxiousafferent input and a stronger pain barrage is transmitted into thebrain. Inhibition of the second phase response is indicative of acentral mechanism of drug action.

The procedure for the formalin test is as follows: male rats are placedin a Plexiglas chamber and observed for 30–45 min. to observe theirbaseline activity. Multiple groups of animals are pretreated with eithervehicle or different doses of a test compound. Animals are dosed withthe drug of interest either 40 min., if by the intraperitoneal route, or90 min., if by the oral route, prior to injection of formalin into ahind paw (under the dorsal skin; 0.05 mL of sterile 5% formalin). Thenumber of paw flinches and licks during first phase (0–5 min.) andsecond phase (20–35 min.) are scored and recorded. Flinch and lickresponses are calculated as percentage of inhibition compared with themean score of a saline control group. Drug potencies are expressed asthe dose which causes 50% of the maximum inhibitory effect, (“ID₅₀”).Student t-tests are used for statistical analysis to determine thesignificance of drug effects. Compounds are considered active based ontheir ability to inhibit the flinch response.

CHEMICAL METHODS

2-hydroxyquinoline and 2-chloroquinoline intermediates of exemplarycompounds disclosed herein, see Table 1, were prepared as describedbelow.

Starting quinoline diols were made using a standard procedure (Fischer,M., R. Laschober, et al. (1996). “3,4,8-Trimethoxy-2-quinolone.Synthesis of a new alkaloid from Eriostemon gardneri.” Sci. Pharm.64(3/4): 353–358; Patel, G. H. and C. M. Mehta (1960). “Synthesis of2,4-Dihydroxyquinolines Using Polyphosphoric Acid as the CyclizingAgent.” J. Sci. Industrial Res. 19B: 436). The methods and subjectmatter of these journal articles are incorporated herein in theirentirety by reference. All other reagents were purchased from Acrosorganics and used directly.

Intermediate 1 6-methoxy-4-(3,4-dichlorophenyl)amino-2-hydroxyquinoline

General procedure 1

6-Methoxyquinolinediol (2.5 g, 13.08 mmol) and 3,4-dichloroaniline (4.2g, 26.25 mmol) were placed in a 100 mL Teflon vessel for use with anEthos 1600 Lab Microwave. NMP was then added (9 mL), followed by HCl indioxane (5 mL, 4 M). The slurry was irradiated to achieve a 200° C.temperature using a 400 W upper setpoint for 30 min. The mixture wascooled, suspended in methanol (20 mL), and filtered to afford 1.049 g ofthe title compound. ¹H NMR (300 MHz, DMSO): 3.84 (s, 3H), 5.84 (s, 1H),7.23 (td, ?H, J=8.88, 8.48 Hz), 7.35(tt, ?H, J=8.88, 2.42 Hz), 7.56(d,?H, J=2.42 Hz), 7.66 (˜s, 2H), 8.71(s, 1H), 11.10(s, 1H).

Intermediate 2 4-(3,4-dichlorophenyl)amino-2-hydroxyquinoline

General procedure 2

2,4-quinolinediol (10.0 g, 62.1 mmol) and 3,4-dichloroaniline (13.1 g,80.7 mmol) were heated to 190° C. in 25 mL NMP for 48 h. After heatingwas discontinued, solids began to precipitate. These were collected,treated with 8.5 mL of HCl in isopropanol and sonicated in a 4:1 mixtureof acetone:isopropanol. After 2.5 h of sonication, the solids werecollected. The sonication cycle was repeated to afford 6.79 g ofmaterial (19.9 mmol, 32%). ¹H NMR (300 MHz, DMSO): 12.7 (s,1 H), 9.34(s, 1 H), 8.41 (d, 1 H, J=9 Hz), 7.71 (m, 3 H), 7.6 (d, 1 H, J=8.1 Hz),7.44 (m, 2 H), 6.25 (s, 1 H), 6.08 (broad s, 1 H).

Intermediate 3 6-Bromo-4-(3,4-dichlorophenyl)amino-2-hydroxyquinoline

Prepared using general procedure 1; 6-Bromo-2,4-quinolinediol (3 g, 12.5mmol), 3,4-dichloroaniline, 6.25 mL of 2 M HCl in Et₂O were irradiatedto 180° C. (400 W max) for 40 min. to afford 1.94 g (5.05 mmol, 40%). ¹HNMR (300 MHz, DMSO): 11.36 (s, 1H), 8.83(s, 1H), 8.28(s, 1H), 7.70(dd,1H, J=8.88, 1.61 Hz), 7.64(d, 1H, J=8.88 Hz), 7.55(d, 1H, J=2.02 Hz),7.33(dd, 1H, J=8.88, 2.42 Hz), 7.25(d, 1H, J=8.88 Hz), 5.86(s, 1H).

Intermediate 4 6-Fluoro-4-(3,4-dichlorophenyl)amino-2-hydroxyquinoline

Prepared using general procedure 1; 6-Fluoro-2,4-dichloroquinolinediol(2.5 g, 13.95 mmol), 3,4-dichloroaniline (4.5 g, 27.91 mmol), 5 mL of 4M HCl in dioxane (20 mmol) were irradiated to 180° C. (400 W max) for 40min. Pouring onto methanol followed by collection of the solids,afforded 2.136 g of material (6.61 mmol, 47%). ¹H NMR (300 MHz, DMSO):11.48(s, 2H), 8.74(s, 1H), 7.92(dd, 1H, J=10.70, 2.60 Hz), 7.65(d, 1H,J=8.88 Hz), 7.56(d, 1H, J=2.42 Hz), 7.49(d, 1H), 7.33(m, 2H), 5.89(s,1H).

Intermediate 5 8-Bromo-2-Hydroxy-4-(2,3-dichlorophenyl)aminoquinoline

Prepared using general procedure 1; 8-Bromo-2,4-quinolinediol (4 g,16.66 mmol), 3,4-dichloroaniline (8 g, 49.38 mmol), 2.78 mL of 6 M HCl(16.68 mmol) and 20 mL NMP were irradiated to achieve 200° C. (400 Wmax) for 30 min. Cooling and precipitation by pouring onto 100 mL waterafforded 3.52 g of product (9.16 mmol, 55%). ¹H NMR (300 MHz, DMSO):9.77(s, 2H), 8.95(s, 1H), 8.11(dd, 1H, J=8.48, 0.81 Hz), 7.89(dd, 1H,J=7.67, 0.81 Hz), 7.66(d, 1H, J=8.88 Hz), 7.58(d, 1H, J=2.42 Hz),7.36(dd, 2H, J=8.48, 2.42 Hz), 7.20(t, 1H, J=8.07 Hz), 5.88(s, 1H).

Intermediate 6 8-Fluoro-2-Hydroxy-4-(2,3-dichlorophenyl)aminoquinoline

Prepared using general procedure 1; 8-Bromo-2,4-quinolinediol (4 g,22.33 mmol), 3,4-dichloroaniline (10.88 g, 66.66 mmol), 3.7 mL of 6 MHCl (22.2 mmol) and 20 mL NMP were irradiated to achieve 200° C. (400 Wmax) for 30 min. Cooling and precipitation by pouring onto 100 mL waterafforded 3.2 g of product (9.90 mmol, 44%). ¹H NMR (300 MHz, DMSO):9.80(s, 2H), 8.94(s, 1H), 8.12(d, 1H, J=8.48 Hz), 7.90(d, 1H, J=7.67Hz), 7.66(d, 1H, J=8.88 Hz), 7.58(d, 1H, J=2.42 Hz), 7.37(dd, 1H,J=8.88, 2.42 Hz), 7.12(t, ?H, J=7.87 Hz), 5.90(s, 1H).

Intermediate 7 8-Bromo-2-chloro-4-(3,4-dichlorophenyl)aminoquinoline

General Procedure 3

8-Bromo-2-hydroxy-4-(3,4-dichlorophenyl)aminoquinoline (3.52 g, 9.16mmol) and 26 mL of POCl₃ were heated to 120° C. for 4 h. The bulk of thePOCl₃ was distilled (˜18 mL), and the reaction cooled. The solution waspoured slowly onto warm water to get a gummy solid. The water wasdecanted and the solids washed with several portions of water. A finalwash with toluene and drying under vacuum afforded 1.43 g of material(3.55 mmol, 38.8%). ¹H NMR (300 MHz, DMSO): 9.58(s, 1H), 8.39(d, 1H,J=8.48 Hz), 8.15(d, 1H, J=7.67 Hz), 7.70(d, 1H, J=8.88 Hz), 7.66(d, 1H,J=2.42 Hz), 7.51(dd, 1H, J=8.48, 7.67 Hz), 7.44(d, 1H, J=8.88, 2.42 Hz),6.90(s, 1H).

Intermediate 8 8-Fluoro-2-chloro-4-(3,4-dichlorophenyl)aminoquinoline

Prepared using general procedure 3;8-Fluoro-2-hydroxy-4-(3,4-dichlorophenyl)aminoquinoline (3.2 g, 9.90mmol) and 23 mL of POCl₃ were heated to 120° C. for 4 h. Isolationafforded 0.69 g of material (1.98 mmol, 20%). ¹H NMR (300 MHz, DMSO):9.50(s, 1H), 8.17(d, ?H, J=7.67 Hz), 8.04(d, 1H, J=5.25 Hz), 7.79(d, 1H,J=5.65 Hz), 7.71(d, 1H, J=8.88 Hz), 7.61(m, 1H), 7.45(dd, 1H, J=8.88,2.42 Hz), 6.88(s, 1H).

Intermediate 9 2-Chloro4-(3,4-dichlorophenyl)aminoquinoline

Prepared using general procedure 3;2-Hydroxy-4-(3,4-dichlorophenyl)aminoquinoline (1.5 g, 4.9 mmol) and 9.2mL of POCl₃ were heated to 120° C. for 4 h. Isolation afforded 1.075 gof material (3.32 mmol, 68%). ¹H NMR (300 MHz, DMSO): 9.61(s, 1H),8.40(d, 1H, J=8.07 Hz), 7.82(m, 2H), 7.65(m, 3H), 7.45(dd, 1H, J=8.68,2.63 Hz), 6.87(s, 1H).

Intermediate 10 6-Bromo-2-chloro-4-(3,4-dichlorophenyl)aminoquinoline

Prepared using general procedure 3;6-Bromo-2-hydroxy-4-(3,4-dichlorophenyl)aminoquinoline (1.94 g, 5.05mmol) and 30 mL of POCl₃ were heated to 120° C. for 4 h. Isolationafforded 1.209 g of material (3.00 mmol, 60%). ¹H NMR (300 MHz, DMSO):9.69(s, 1H), 8.71(s, 1H), 7.91(d, 1H, J=8.88 Hz), 7.78(d, 1H, J=8.88Hz), 7.69(d, 1H, J=8.88 Hz), 7.68(s, 1H), 7.44(dd, 1H, J=8.68, 2.22 Hz),6.91(m, 1H).

Intermediate 11 6-Fluoro-2-chloro-4-(3,4-dichlorophenyl)aminoquinoline

Prepared using general procedure 3;6-Fluoro-2-hydroxy-4-(3,4-dichlorophenyl)aminoquinoline (2.136 g, 6.6mmol) and 30 mL of POCl₃ were heated to 120° C. for 4 h. Isolationafforded 0.654 g of material (1.914 mmol, 29%). ¹H NMR (300 MHz, DMSO):9.55(s, 1H), 8.27(dd, 1H, J=10.50, 2.02 Hz), 7.92(dd, 1H, J=8.88, 5.65Hz), 7.70(m, 3H), 7.45(dd, 1H, J=8.48, 2.02 Hz), 6.90(s, 1H).

Intermediate 12 6-Methoxy-2-chloro-4-(3,4-dichlorophenyl)aminoquinoline

Prepared using general procedure 3;6-Methoxy-2-hydroxy-4-(3,4-dichlorophenyl)aminoquinoline (1.05 g, 3.13mmol) and 26 mL of POCl₃ were heated to 120° C. for 4 h. Isolationafforded 0.685 g of material (1.94 mmol, 61%).

Intermediate 13 4-Chloro-2-(N-2-(4-morpholino)phenylamino)quinoline

2,4-Dichloroquinoline (300 mg, 1.51 mmol), 2-morpholinoaniline (297 mg,1.66 mmol), 1 M HCl in Et₂O (3 mL) and NMP (3 mL) were added to a 10 mLSchlenk Flask. The ether was removed with a stream of nitrogen, and theflask heated to 130° C. for 16 h. The reaction was cooled and passedthrough a 500 mg tC18 Sep Pak© (Waters Corp), and purified on 3×100Novapak HR RCM segments using methanol/water/0.1% TFA at 25 mL per min.Evaporation of the solvents afforded 326.2 mg of product as a TFA salt(0.718 mmol, 48%) along with the product arising from bis-addition (116mg, 0.22 mmol, 15%). Bis-addition ¹H NMR (DMSO, 300 mHz): 12.31(s, 1H),9.90(s, 1H), 9.70(s, 1H), 8.49 (d, 1H, J=8.48 Hz), 7.84 (m, 2H), 7.57(m, 1H), 7.33 (m, 4H), 7.14 (m, 4H), 5.85 (s, 1H), 3.38(s, 8H), 2.87 (s,4H).

Intermediate 14 4-(2-methoxyethylamino)-2-hydroxyquinoline

Prepared as in general procedure 1; Quinoline diol (1 g, 6.2 mmol),2-methoxyethylamine (700 mg, 9.3 mmol) afforded 722 mg of product (53%)which was taken on directly.

Intermediate 15 2-Chloro-4-(2-methoxyethylamino)quinoline

Prepared using general procedure 3;4-(2-methoxyethylamino)-2-hydroxyquinoline (611 mg, 2.8 mmol) and 2.5 mLof POCl₃ were heated to 120° C. for 8 days. Isolation afforded 610 mg ofmaterial (92%).

Diversification Library: General Procedure 4

2-Chloroquinoline intermediates (prepared above) (300 mg) were coupledwith 2 equivalents of amine (from Table I) using a ChemSpeed robot. Eachcoupling was carried out in NMP for the time and temperature indicatedin Table 2, and the isolated yield was calculated for >50% of theentries. The compounds were characterized with HPLC, HPLC-MS andpurified by preparative HPLC.

TABLE 1 Amine reagents used in synthesis of compounds of the presentinvention. A 2-hydroxyaniline B 2-methoxyaniline C 2-methylaniline D2-aminoaniline E 2-fluoroaniline F Aniline G 3,4-dichloroaniline H4-fluoro-3-chloroaniline J 3,4-dimethoxyamine L 2-morpholinoaniline M6-aminobenzothiazole N 4-fluoroaniline O 2-chloroaniline

EXAMPLES

Exemplary compounds 1 to 34 inclusive are illustrated in Table 2 whichshows the name of each compound and the specifics of reaction time,reaction temperature, yield and molecular ion. Quinoline precursors wereprepared by the general procedures 1, 2 and 3 as appropriate, reactedwith an amine from Table 1 as indicated, using general procedure 4.

TABLE 2 Physical data for Examples 1–34. MS Ex. # amine Compound Nameyield time temp. (m + H) 1 L N2,N4-bis-(2-morpholin-4-yl-phenyl)- 2917:00 130 482 quinoline-2,4-diamine 2 L N4-(3,4-Dichloro-phenyl)-N2-(2-74 24:00 107 465 morpholin-4-yl-phenyl)-quinoline-2,4- diamine 3 B8-Bromo-N4-(3,4-dichloro-phenyl)- 45 16:00 180 488N2-(2-methoxy-phenyl)-quinoline-2,4- diamine 4 C8-Bromo-N4-(3,4-dichloro-phenyl)- 36 16:00 180 472N2-o-tolyl-quinoline-2,4-diamine 5 E8-Bromo-N4-(3,4-dichloro-phenyl)-N2- 76 16:00 180 476(2-fluoro-phenyl)-quinoline-2,4- diamine 6 O8-Bromo-N2-(2-chloro-phenyl)-N4- 58 16:00 180 492(3,4-dichloro-phenyl)-quinoline-2,4- diamine 7 A2-[8-Bromo-4-(3,4-dichloro- 46 16:00 180 474phenylamino)-quinolin-2-ylamino]- phenol 8 DN2-(2-Amino-phenyl)-8-bromo-N4- 34 16:00 180 473(3,4-dichloro-phenyl)-quinoline-2,4- diamine 9 A2-[4-(3,4-Dichloro-phenylamino)- 56 18:00 150 396quinolin-2-ylamino]-phenol 10 B N4-(3,4-Dichloro-phenyl)-N2-(2- 54 18:00150 410 methoxy-phenyl)-quinoline-2,4- diamine 11 CN4-(3,4-Dichloro-phenyl)-N2-o-tolyl- 59 18:00 150 394quinoline-2,4-diamine 12 A 2-[4-(3,4-Dichloro-phenylamino)-8- 54 16:00180 414 fluoro-quinolin-2-ylamino]-phenol 13 BN4-(3,4-Dichloro-phenyl)-8-fluoro-N2- 36 16:00 180 428(2-methoxy-phenyl)-quinoline-2,4- diamine 14 DN2-(2-Amino-phenyl)-N4-(3,4- 54 16:00 180 413dichloro-phenyl)-8-fluoro-quinoline- 2,4-diamine 15 FN4-(3,4-Dichloro-phenyl)-8-fluoro-N2- 32 16:00 180 398phenyl-quinoline-2,4-diamine 16 E N4-(3,4-dichloro-phenyl)-8-fluoro-N2-22 16:00 180 416 (2-fluoro-phenyl)-quinoline-2,4- diamine 17 C6-bromo-N4-(3,4-dichloro-phenyl)-N2- 57 16:00 150 472o-tolyl-quinoline-2,4-diamine 18 D N2-(2-amino-phenyl)-6-bromo-N4- 6516:00 150 473 (3,4-dichloro-phenyl)-quinoline-2,4- diamine 19 E6-bromo-N4-(3,4-dichloro-phenyl)-N2- 67 16:00 150 476(2-fluoro-phenyl)-quinoline-2,4- diamine 20 F6-bromo-N4-(3,4-dichloro-phenyl)-N2- 89 16:00 150 458phenyl-quinoline-2,4-diamine 21 A 2-[4-(3,4-dichloro-phenylamino)-6- 6516:00 150 414 fluoro-quinolin-2-ylamino]-phenol 22 EN4-(3,4-dichloro-phenyl)-6-fluoro-N2- 36 16:00 150 416(2-fluoro-phenyl)-quinoline-2,4- diamine 23 FN4-(3,4-dichloro-phenyl)-6-fluoro-N2- 68 16:00 150 398phenyl-quinoline-2,4-diamine 24 B N4-(3,4-dichloro-phenyl)-6-methoxy- 1216:00 150 440 N2-(2-methoxy-phenyl)-quinoline-2,4- diamine 25 CN4-(3,4-dichloro-phenyl)-6-methoxy- 30 16:00 150 424N2-o-tolyl-quinoline-2,4-diamine 26 B6-bromo-N4-(3,4-dichloro-phenyl)-N2- 27 16:00 105 488(2-methoxy-phenyl)-quinoline-2,4- diamine 27 DN2-(2-amino-phenyl)-N4-(3,4-dichloro- 34 16:00 150 395phenyl)-quinoline-2,4-diamine 28 EN4-(3,4-dichloro-phenyl)-N2-(2-fluoro- 65 16:00 150 398phenyl)-quinoline-2,4-diamine 29 F N4-(3,4-dichloro-phenyl)-N2-phenyl-48 16:00 150 380 quinoline-2,4-diamine 30 A 2-[6-bromo-4-(3,4-dichloro-82 16:00 150 474 phenylamino)-quinolin-2-ylamino]- phenol 31 F8-bromo-N4-(3,4-dichloro-phenyl)-N2- 15 16:00 180 458phenyl-quinoline-2,4-diamine 32 D N2-(2-amino-phenyl)-N4-(3,4- 45 16:00150 425 dichloro-phenyl)-6-methoxy-quinoline- 2,4-diamine 33 EN4-(3,4-dichloro-phenyl)-N2-(2-fluoro- 63 16:00 150 428phenyl)-6-methoxy-quinoline-2,4- diamine 34 FN4-(3,4-dichloro-phenyl)-6-methoxy- 35 16:00 150 410N2-phenyl-quinoline-2,4-diamineBiological data for selected compounds is shown in Table 3.

TABLE 3 Biological Data Formalin test % inhibition FLIPr Ex. # CompoundStructure (dose, time route) IC₅₀ (N)  2

42 (60, 40, IP) 10.00 14

4.01 27

9.82 33

10.00

1. A compound of formula I:

wherein: A is phenyl or morpholine; R¹ at each occurrence isindependently selected from halogen, (C₁–C₆)alkyl, heterocyclyl, OH,(C₁–C₆) alkoxy or NR² ₂; b is an integer selected from 0, 1, 2 or 3; R²at each occurrence is independently selected from H or (C₁–C₄)alkyl; R³at each occurrence is independently selected from halogen or(C₁–C₄)alkyl; d is an integer selected from 0, 1, 2 or 3; R⁴ is selectedfrom H or (C₁–C₄)alkyl; R⁵ is selected from the group consisting of H,halogen, (C₁–C₃)alkyl, (C₁–C₃)perfluoroalkyl, (C₁–C₃)alkoxy, hydroxy,NH₂ or NHR²; R⁶ is selected from H, halogen, (C₁–C₆)alkyl, (C₁–C₄)perfluoroalkyl, (C₁–C₆)alkoxy, hydroxy, (C₁–C₆)alkanoyl, C(═O)NR² ₂ or—NR² ₂; and R⁷ selected from H or methyl.
 2. A compound of claim 1wherein: R⁴ and R⁷ are H; and R⁵ is selected from H, halogen,(C₁–C₃)alkyl, (C₁–C₃)perfluoroalkyl or (C₁–C₃)alkoxy.
 3. A method forthe treatment of pain in a subject suffering therefrom, comprisingadministering to said subject a pain-ameliorating effective amount of acompound according to claim
 1. 4. A pharmaceutical compositioncomprising a therapeutically-effective amount of a compound according toclaim 1 together with at least one pharmaceutically-acceptable excipientor diluent.
 5. A method for preparing a compound according to claim 1,said method comprising: a) reacting a substituted 2,4-quinolinediol(formula II) with three equivalents of an aryl amine inN-methylpyrrolidinone and 6N HCl in 2-propanol, in a sealed tube at atemperature of about 180° C.;

 wherein R³, R⁴, R⁵, R⁶ and d are as defined as in claim 1; b)chlorinating the 2-hydroxy-4-aminoquinoline compound according toformula III by refluxing with POCl₃ to form a compound according toformula IV;

 wherein R³, R⁴, R⁵, R⁶ and d are as defined as in claim 1; and c)reacting the 2-chloro-4-aminoquinoline compound with a compound havingthe formula R⁷—NH—A—(R¹)b in N-methylpyrrolidinone at a temperature of100–180° C.;

 wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, A, b, and d are as defined as inclaim
 1. 6. The method according to claim 5, wherein step c) isperformed in a parallel fashion using robotic instrumentality.
 7. Amethod for preparing a compound according to claim 1, said methodcomprising: a) reacting a substituted 2,4-quinolinediol in accord withformula II with two equivalents of an aryl amine inN-methylpyrrolidinone and 4 M HCl in dioxane, in a sealed tube usingmicrowave irradiation at a 400 W upper set point to maintain a 200° C.temperature for 30 minutes;

 wherein R³, R⁴, R⁵, R⁶ and d are as defined as in claim 1; b)chlorinating the 2-hydroxy-4-aminoquinoline compound according toformula III by refluxing with POCl₃ to form a compound according toformula IV;

 wherein R³, R⁴, R⁵, R⁶ and d are as defined as in claim 1; and c)reacting the 2-chloro-4-aminoquinoline compound with a compound havingthe formula R⁷—NH—A—(R¹)_(b) in N-methylpyrrolidinone at a temperatureof 100–180° C.;

 wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, A, b, and d are as defined as inclaim
 1. 8. The method according to claim 7, wherein step c) isperformed in a parallel fashion using robotic instrumentality.